The protective effect of Azadirachta indica (neem) against metabolic syndrome: A review

Metabolic syndrome is a condition associated with obesity, diabetes, dyslipidemia, and high blood pressure. Recently, the use of phytochemicals is suggested in the control and treatment of metabolic syndrome. The Azadirachta indica (neem) is an evergreen tree belonging to the family of Meliaceae. Multiple studies have been confirmed the anti-diabetic and anti-hypertension, anti-hyperlipidemia, and anti-obesity effects of neem. In this review, we reported the protective effects of neem against the complications of metabolic syndrome with a special focus on mechanisms that are involved. It has been shown that neem can control hyperglycemia and hypertension through over-expression of transcription factor nuclear factor erythroid 2–related factor 2 (Nrf2) and anti-oxidant effects. Neem also reduced the glucose uptake through up-regulation of glucose transporter 4 (GLUT4) and inhibition of key intestinal enzymes such as glucosidases. Moreover, neem showed anti-hypertensive effects possibility via the block of calcium channels, up-regulation of endothelial nitric oxide synthase (eNOS), and extracellular signal-regulated kinases 1/2 (ERK1/2) signaling pathway. Anti-oxidant effects play an important role in protective mechanisms of neem against metabolic syndrome and its complications.


Introduction
Metabolic syndrome (MetS) is a common metabolic disorder that is described for more than multiple decades. The MetS is also known as insulin resistance syndrome and syndrome X (1). Physical inactivity, smoking, increasing age, obesity, and positive family history are risk factors associated with its development (2). Epidemiologic data have been suggested that the prevalence of MetS among the population over 60 years is the highest, and it is increasing among children and adolescents (3). People with MetS have a higher risk of type 2 diabetes and cardiovascular disease (CVD) (4,5). Also, hypertension and an increase in triglyceride (TG)/ high-density lipoprotein (HDL) cholesterol ratio are important components of the MetS and are one of the most risk factors for CVD (6,7).
Moreover, several studies have been shown other side effects of MetS such as fatty liver disease (8), cirrhosis (9), and polycystic ovary syndrome (10). The pathogenesis of MetS has not been clearly defined, but insulin resistance, oxidative stress, and chronic inflammation are key pathogenic factors of it. Insulin resistance has a role in the development of diabetes mellitus. It has been reported that oxidative stress accelerates the development of complications of MetS. The activation of the inflammatory pathway leads to insulin resistance and diabetes (11,12).
The first-line treatment of MetS is lifestyle modification on diet, weight, and physical activity. Second-line therapy for patients with MetS is drug therapy (13). In line with the treatment of MetS, the use of herbs has been regarded. Medicinal plants contain bioactive compounds with various metabolic effects.
In several studies have been reported the protective potential of plants and herbs against MetS such as Capsicum annuum L. (14), Crataegus pinnatifida (15), and green tea (16). Taken together, the management of complications of MetS is the aim of the treatment in these patients, and medicinal plants can play an important role in its treatment. Neem (Azadirachta indica) is an evergreen tree of southeastern Asia that is widely distributed in the Indian subcontinent. The height of this tree is approximately 15-20 m and sometimes even up to 35-40 m. The word A. indica was derived from the Persian language. The Azad means "free," and the dirakht is meaning "tree," and "I" refer to "Indian origin." (17). Neem is a common name, and also it's known with the name of Nimbay, Veppai, Ariyaveppu, Vepa in India (18). More than 300 compounds are derived from different parts of neem, such as leaves, flower, seed, fruit, bark, and root. Nonisoprenoids and isoprenoids metabolites are two major groups of these compounds. Some active constituents of neem include nimbidin, sodium nimbidate, nimbin, nimbolide, gallic acid, azadirachtin, and polysaccharides (19). Nimbidin, as a major constituent extracted from

Effects of neem on metabolic syndrome Effects of neem on high blood pressure
One of the main constituents of MetS is high blood pressure (BP). Effects of several plants investigated on BP such as Aloe vera (25) and Capsicum annuum L. (14). High BP has an increased risk of heart and blood vessel diseases. Multiple mechanisms induce high BP, including: (1) Calcium channels initiate vascular smooth muscle contraction through the release of calcium, which is mediated by Ca 2+ influx via L-type and voltage-gated calcium channels. Calcium channels have an important role in the induction of high BP (26); (2) the extracellular signal-regulated kinase (ERK 1 and 2) are from the mitogen-activated protein kinases (MAPK) family. ERK1 and ERK2 play an essential role in the regulation of vascular smooth muscle contraction. Down-regulation of ERK 1 and 2 genes reduces both vascular smooth muscle cell growth and vasoconstriction. Therefore, ERK 1and 2 are a target for the induction of high BP (27); (3) Nitric oxide (NO) is a vasodilator produced by nitric oxide synthase (NOS) enzymes. The NOS isozymes and NO level are candidates for involvement in high BP (28); (4) Nuclear factor erythroid 2-related factor 2 (Nrf2) as a transcription factor involves transcriptional induction of several anti-oxidant genes. Nrf2 regulates signaling pathway functions to reduce reactive oxygen radicals (ROS) production. The down-regulation of Nrf2 expressions induces ROS production and resulting in high BP. The contraction of smooth muscle and depletion of NO are the mechanisms of ROS-induced high BP (29). Several studies have been reported the beneficial effects of different extracts (aqueous, alcoholic) of neem leaves against high BP which have been categorized in Table  1. The mechanisms underlying the protective effect of neem against high BP have been presented in Figure  1. The mechanisms are including the block of calcium channels (30), up-regulation of ERK 1 and 2 (31) and Nrf2 gene expression, reduction of oxidative stress markers, and elevation of the nitric oxide (NO) levels (32). It has been reported that neem exerted the vasodilatation effects possibility through the block of calcium channel in the isolated aorta of rat and rabbit. Also, it has been shown that neem exerted dose-dependent fall in arterial pressure of isolated guinea-pig atrial (30). The downregulation of ERK1 and 2 have been reported in cardiac and renal tissues of rats treated by sodium fluoride (600 ppm in drinking water). Neem protected hypertensive rats through (100 and 200 mg/kg, p.o.) up-regulation of ERK (31). L-NAME (N ω -Nitro-L-Arginine Methyl Ester) is a NOS inhibitor and reduces NO bioavailability. Polyphenol-rich fraction of neem (100 and 200 mg/ kg) restored NO level in rats were treated with L-NAME 1 Figure 1. Main mechanisms of neem on high blood pressure. Mechanisms underlying the protective effect of neem against high BP are including the block of calcium channels, up-regulation of ERK 1/2 and Nrf2 gene expression, and normalize serum of NO bioavailability. ARE: Anti-oxidant response element; eNOS: endothelial nitric oxide synthase; ERKs: extracellular-regulated kinases; GPX: glutathione peroxidase; GST: glutathione S-transferases, Nrf2: nuclear factor erythroid-2-related factor 2; NO: nitric oxide; ROS: reactive oxygen species; SOD: superoxide dismutase (orally, 40 mg/kg) (32). The methanol extract of neem (orally, 100 and 200 mg/kg for 7 days) increased NO level in serum of rats exposed to sodium fluoride (NaF) (600 ppm in drinking water) (31). The crude (0.3-3 mg/ ml), aqueous (1-5 mg/ml) and ethyl acetate (0.1-1 mg/ kg) extracts of neem induced endothelium-dependent vasorelaxation in isolated rat aorta (30).
A study has been reported on the effect of neem leaves on high BP of the 90 diabetic patients aged 40-60, which were kept under observation for a month. During the study, patients received 2 g powder of neem daily for three months. A significant reduction was observed in the BP of treated patients (34).

Effects of neem on hyperlipidemia
One of the most components of MetS is hyperlipidemia. Many medicinal plants showed positive effects on hyperlipidemia such as barberry (Berberis vulgaris) (35) and rosemary (Rosmarinus officinalis) (36). Plasma lipid levels elevate in people with diabetes and obesity (37). Hyperlipidemia contributes to impair endothelial function, development of atherosclerosis, and coronary heart disease (CHD) through the enhancement of oxidative stress (38). Antioxidant defense system (SOD and GPx) protects plasma lipoproteins against oxidative stress (39). The elevation of ROS generation under stress conditions (diabetes and obesity) causes oxidative damage of lipoproteins in the plasma (40,41). The oxidation of lipoproteins increases TG, very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL) concentrations in plasma (40). The measure of serum TG, total cholesterol (TC), LDL, HDL, and cholesterol is the reference procedure for the determination of lipid profile (42). As mentioned in Table 2 several studies have been shown the effects of neem in the management of hyperlipidemia.

Effects of neem on obesity
Obesity and overweight are a serious health problem that is increasing worldwide. Obesity is associated with a life expectancy decrease and a significant increase in mortality (56). Stress, inadequate sleep, intake of alcohol, inactivity, unhealthy diet, age, genetic are some of the risk factors for obesity Figure 2 (57). Diabetes, heart disease, high blood pressure, hyperlipidemia, and atherosclerosis are obesity-related complications (58).
Lipase and α-glucosidase are two types of obesity agents that use of their inhibitors can be the ideal therapy for obesity control (59,60). The aqueous and methanolic extract of stem bark and roots of neem (520 µg/ml) inhibited pancreatic lipase and α-glucosidase in an in vitro system (59). But, the leaf extract of neem as a medicinal plant (500 mg/kg, orally) is not decreased the body weight in rats were treated for 28 days (60). There are a few studies available on the protective effects of neem against obesity which has been included in Table 3. Therefore, the effects of neem on obesity cannot be appropriately explained, and further studies are needed.

Effects of neem on diabetes
Diabetes, as a growing public health problem, is characterized by impairment of systemic insulin secretion, reduction of insulin action, and resulting in hyperglycemia (61). Diabetes-associated main complications are nerve damage (62), myocardial infarction (63), atherosclerosis (64), renal failure (65), blindness (66), and limb amputation (67). The microvascular disease has been known as the foremost cause of these complications (68). Glucose-mediated vascular damage occurs as a result of the overproduction of ROS and oxidative stress (69). Enzymatic and nonenzymatic anti-oxidants are defense mechanisms against oxidative stress. Common enzymatic antioxidants include SOD, CAT, GPx, and glutaredoxin (GRx). Vitamins A, C, E, and glutathione are common nonenzymatic anti-oxidants (70). Pancreatic beta cells are more susceptible to oxidative stress than other cells because they have relatively low levels of anti-oxidants (71). Therefore, the reduction of ROS and induction of anti-oxidant activity are therapeutic approaches to decrease hyperglycemia and diabetes (72). On the other hand, salivary α-amylase and intestinal glucosidases play an essential role in the digestion of starch to produce glucose in the small intestine (73). Also, the inhibition of these enzymes could be effective in the control of diabetes (74). Several reports have been shown that medicinal plants useful for the management and treatment of diabetes. Some of these plants include Vernonia amygdalina (75), Nigella sativa L. (76), grapes (Vitis vinifera) (77), and Allium sativum (garlic) (78) which are useful in the remedy of diabetes. The use of the neem is most popular to control diabetes in different regions of the world, such as India (79), Pakistan (80), Bengal (81), Indonesia (82), and Northwest Nigeria (83). Glucagon-like peptide-1 (GLP-1) is a hormone that plays an essential role in the release of insulin and is inactivated by dipeptidyl peptidase IV (DPP-IV) (84,85). Inhibition of DPP-IV (a peptidase) is a method for diabetes treatment (86). In this method, substrate Gly-Pro-p-Nitroanilide (GPPN) was cleaved to paranitroanilide (a yellow-colored product) by DPP-IV and the absorbance was measured at 380 nm. Inhibitory activity of neem leaves (35 μl with varying concentrations) was determined on DPP-IV activity via this method, and neem exhibited a weak inhibitory activity (17%) on DPP-IV (82).

• Effects of neem in diabetic human
Neem is available as a dietary supplement in an herbal mixture in North America. Treatment with this dietary supplement (2 capsules 3 times per day) for 3 months in type 2 diabetic patients (the ages of 18 and 70) improved glucose control and HbA1c levels (87). The study of Kochhar has been investigated the antidiabetic effect of neem in 90 diabetic men 40 to 60 years of age. Subjects received 2 g of neem leaf powder daily for three months. The results of this study showed that neem reduces sweating, headache, burning feet, itching, polydipsia, and polyphagia in diabetic humans (34).

• Effects of neem in alloxan/streptozotocin-induced diabetic animals •• Effects of neem in alloxan-induced diabetic rats
Alloxan is a toxic glucose analog that accumulates in pancreatic beta cells via glucose transporter 2 (GLUT2) Stem-bark and roots/ aqueous and methanolic IC50: 520 µg/ml In vitro Inhibited pancreatic lipase and α-glucosidase (59) and inhibits its function. The intraperitoneal injection of alloxan (at doses of 100, 120, and 150 mg/kg) is a conventional method for the induction of diabetes in rat models (88). The oral administration of ethanolic extract of neem in different doses (100 to 800 mg/ kg for 14 or 28 days) reduced blood glucose levels in rats which were treated with alloxan (52,89,90). The combination of neem (50 mg/kg) with Gynura procumbens ethanolic (112.5 mg/kg) extracts (2 times a day for 15 days) increased insulin expression, decreased blood glucose concentration, and improved the morphology of the islets of Langerhans and beta-cells in rats (91). The aqueous extract of neem leaf and bark was effective in reducing oxidative stress markers and lipid peroxidation of the blood sample, liver, and kidney tissues in diabetic rats (92,93). Polyherbal formulation (PHF) is containing more than one herb that is used all around the world to treat diseases (94). PHFs used in the treatment of diabetes are including Karnim Plus and DIA7. The antidiabetic activity of Karnim Plus and DIA7 is investigated in rats treated with alloxan. Karnim Plus and DIA7 contain neem extract and decrease blood glucose levels in diabetic rats (51,95). In Table 4, different studies on the effect of neem on diabetes have been summarized.

•• Effects of neem in streptozotocin-induced diabetic mice
The decrease of serum glucose levels and increase of glycogen content, plasma insulin, and c-peptide levels with aqueous extract of neem have been shown in mice treated with STZ (110,111). Also, the increase of glucose-6-phosphate dehydrogenase (G6PD) activity with neem has been shown in diabetic mice (110). The chloroform extract of neem in addition to reducing glucose level and induction of insulin level decreased oxidative stress markers and LPO in mice treated with STZ-nicotinamide. Also, it reduced glucose-6phosphatase-α (G6Pase), glucokinase (GK), α-amylase, and α-glucosidase activities, and induced HK activity (50). Dianex, an herbal formulation is containing neem at a dose of 7.5 mg/kg. Dianex has been shown hypoglycemic effects in STZ-diabetic mice (49).

• Effects of neem in glucose-induced diabetic animals
The aqueous and ethanolic extracts of neem leaves decreased glucose level and increased insulin secretion in rats were treated with glucose (3 mg/ml, p.o.) (112,113). Also, the potential use of ethanolic extract rhizome of neem (300 mg/kg, p.o.) was investigated in mice that were treated with glucose (1 g/kg). In this study, neem reduced blood glucose (114).

Conclusion
In summary, A. indica (neem) is effective in MetS and anti-oxidant effects appear to play an important role in protective mechanisms of neem against MetS and the complications associated with it. Neem increases the expression of Nrf2-mediated anti-oxidant enzymes and can regulate blood pressure and lipid profile. Also, neem inhibits vascular smooth muscle contraction through the block of calcium channels and decreases high blood pressure. Neem up-regulates eNOS expression as a vasodilator and increases NO level. Moreover, neem reduces vasoconstriction through the regulation of the ERK1/2 signaling pathway. In the diabetic condition, neem up-regulates GLUT4 and reduces the glucose uptake. Neem also inhibits intestinal enzymes such as glucosidases. Understanding the signaling pathways help to expand the use of neem in the treatment of the MetS. However, few studies have been conducted to investigate the anti-diabetic, anti-hypertension, antihyperlipidemia, and anti-obesity activities of neem in humans. Therefore, further clinical studies are needed to assess the protective effects of neem.